The NIMH transgenic core facility has several major functions: 1) to produce transgenics for neuroscience research, 2) support research with associated techniques in genetic research in neuroscience, 3) develop new transgenic techniques and model systems and 4) engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Metrics of production over the past year included: a) 12 transgenic mouse projects produced by oocyte injection, with multiple lines produced for each project. b) Ten transgenic rat projects produced by oocyte injection, with multiple lines produced for each project. c) Seven mouse projects first altering the genes of embryonic stem (ES) cells, and then using those to produce mice. 2) Technical Support a) 96 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 25 lines have been re-derived, by transferring lines from pathogen bearing animals into those with defined health status. c) Transgenic project design and assistance have continued to be significant to NIH neuroscience labs without experience in producing transgenic animals. 3) Technical development a) Nuclease mediated genetic engineering: Over the last year the core facility has worked on several projects that use the CRISPR/Cas9 system to modify genes in cells and in animals. In collaboration with labs at the National Insitute on Drug Abuse (NIDA), we have investigated the function of different iterations of the CRISPR/Cas9 system in rats. With Nick Ryba in the National Institute of Dental and Craniofacial Research (NIDCR), we have worked on mice, and with Guoping Feng of the McGovern Institute at Massachusetts Institute of Technology (MIT) we have targeted genes in marmosets. Using this system across species has given the core the opportunity to generalize these methods. This includes optimizing concentration of the injected material, the site of injection in embryos, and the construction of targeting plasmids. b) Transgenic marmosets: The core's collaboration with Erika Sasaki at the Central Institute for Experimental Animals in Kawasaki, Japan continue this year. Methods for harvesting oocytes, in vitro maturation and fertilization are based with those used in Dr Sasaki's laboratory. Cooperation between the Core and Afonso Silva's laboratory in the National Institute of Neurological Disorders and Stroke (NINDS) also continues in an effort to produce marmosets that express the genetically encoded calcium indicator (GECI). Most recently a project using CRISPR/Cas9 technologies to targeted behaviorally relevant genes in marmoset oocytes (see below under collaboration). c) Rat ES lines: Rat ES lines, some of which ubiquitously express the orange fluorescent protein (OFP) have been created in the lab. These lines from Long Evans rats have been cultured for several passages, still express OFP and maintain a morphology that is representative of ES cells. Especially with rat ES lines this is not enough to insure that these lines will contribute to a chimeric animal. The OFP marker will allow the potential contribution of these ES cells to the different tissues of the chimeric animals, as well as germline transmission to offspring of these founder animals. d) Transgenic rat production: in collaboration with NIDA, the core produces transgenic rat lines that are designed in conjunction with transiently delivered transgenes to express genes in discrete populations of central nervous system neurons. These lines are produced in the core facility and then screened for useful expression patterns in NIDA laboratories. This year, four of these lines have been characterized to the point where they are in use in laboratories in NIMH, NINDS and NIDA. We have been collaborating to use the CRISPR/Cas9 system to target specific genes as well as to increase the efficiency of targeting with replacement sequences directed to specific genetic loci. e) Support techniques: several techniques are under development to increase the capacity of the core's support functions. Freezing mouse sperm and improving IVF by using newer methods is a major effort. Freezing rat sperm and completing IVF at an acceptable level is a challenging task in all laboratories, but having consulted with investigators in Japan and Spain, these methods will be improved. 4) Collaborative projects: Targeting an autism gene in non-human primates: Autism spectrum disorders have been linked to several genes but Phelan-McDermid Syndrome has been most closely associated with a single gene: SHANK3. Though other genes in the telomeric region of chromosome 22q my also be involved in creating or effecting the symptoms of the disease. The protein coded by SHANK3 is a component of the post synaptic density (PSD) and incorporates multiple structural motifs that bind other PSD proteins (SH3, PDZ, ankrin, Homer-binding regions). These features indicate that SHANK3 plays a role in the structure as well as the function of the synapse. In mice the inactivation of Shank3 causes deficits in social and repetitive behaviors as well as electrophysiological and circuit disruptions (Peca et al. Nature 472:437, 2011). Rodent models, including the one cited above have been useful in studying the conserved behaviors and functions of mammals, but the spectrum of autism diseases have a range that is lost in the translation of experimental results from rodents to primates. To build on their work in rodents, Guoping Feng's laboratory at the McGovern Institute at the MIT has developed a nuclease-mediated targeting CRISPR/Cas9 system that we have injected into marmoset oocytes. These oocytes have been harvested from the ovary of superovulated donors and allowed to mature in vitro to a specific developmental stage, been fertilized in vitro (IVF), injected with the targeting guide RNAs and the nuclease that specifically cuts the Shank3 locus. These embryos have been analyzed, and the SHANK3 locus is indeed disrupted. Learning and memory: The effect of specific and tightly controlled protein synthesis on learning and memory was studied. In addition, transgenic mouse models have been used to show the role of specific peptide-expressing cells to influence the link between fear and behavior and learning. Manipulating circuitry: Mice have been produced for two separate laboratories which have specific neurons that could be rendered transiently inactive by light activated ion channels. Those laboratories are investigating different neural circuits that are active in learning and addiction. Addictive and reward behavior: Lines of transgenic rats that express GFP in response to afferent input activation of the fos gene were generated in the core facility. These rats are being used by Bruce Hopes laboratory in NIDA to study patterns of neural activity in response to addictive drugs and most recently in the role of stress in reducing the re-establishment of rewarded behavior. mRNA trafficking in neurons: An RNA stem loop structure is necessary for the translocation of message to specific cell compartments of the neuron. Mice that overexpress mRNA with this structure have been produced in an effort to disrupt this translocation machinery. By expressing this transgenic mRNA in different neuronal subtypes, the role for this mechanism for normal function is being studied. In addition, this mechanism could be useful to target specific messages specifically to the synapse.